Antibodies to a fibrinogen binding protein of staphylococcus epidermidis

ABSTRACT

A new fibrinogen binding protein or polypeptide originating from coagulase negative  staphylococci,  biotechnological methods for producing the protein or polypeptide having fibrinogen binding activity and a recombinant DNA molecule coding for the protein (or fragments thereof), and micro-organisms (including viruses) containing this recombinant DNA molecule. The present invention further comprises the therapeutic and diagnostic use of the protein and/or DNA, e.g., a diagnostic kit for determining the presence and/or type of coagulase negative  staphylococci  and a vaccine composition, comprising the protein or DNA.

This application is a divisional application of application Ser. No.09/147,405, filed on Apr. 1, 1999 (of which the entire disclosure of theprior application is hereby incorporated by reference) which is now U.S.Pat. No. 6,733,758, which is a 371 of PCT/SE97/01091, filed on Jun. 18,1997.

The invention relates to the field of gene technology and is concernedwith recombinant DNA molecules, which contain a nucleotide sequencecoding for a protein or polypeptide having fibrinogen-binding activity.Moreover the invention comprises micro-organisms including viruses)containing the aforesaid molecules, and the use thereof in theproduction of the aforesaid protein or polypeptide and their use inbiotechnology. Further, the present invention comprises diagnostic andtherapeutic uses of said new protein, e.g. compositions for activeand/or passive immunisation.

BACKGROUND OF THE INVENTION

During the last decade, the coagulase-negative staphylococci (CNS) haveattracted an increasing attention. Along with the development of humanand veterinary medicine, the number of susceptible hosts have increased.Advanced surgery, an increased use of bio-materials, medication withcytostatics, antibiotics and other drugs together with an increasedfrequency of antibiotica resistant strains of CNS have increased thesusceptibility of the host. Concerning the veterinary importance of theCNS it is known that they can cause e.g. both sub-clinical and clinicalinflammation in the bovine udder. The existence of bacteria that bindspecifically to fibrinogen has been known for many years. The role offibrinogen binding in the interaction process between the host andStaphylococcus aureus is still not clear but the fibrinogen-binding hasbeen considered as one potential virulence factor of this species forinstance in endocarditis (Moreillon et al 1995). No protein withfibrinogen binding properties has hitherto been described originatingfrom CNS. However, the present invention describes the characterizationand isolation of such protein using gene cloning. Furthermore, theinvention describes different methods to measure the fibrinogen bindingactivity on cells of CNS and the use of this protein in biotechnology.

Generally, it might be difficult to obtain a homogeneous and areproducible product if such a binding protein was prepared fromstaphylococcal cells directly. Moreover staphylococci are pathogenic andneed complex culture media, which involves complications in large-scalecultures. There is thus a need for a new method for producing afibrinogen binding protein (or fragments thereof).

SUMMARY OF THE INVENTION

The present invention discloses a new fibrinogen binding protein calledFIG, a DNA molecule encoding said protein and applications for theiruse, according to the attached claims. Importantly, the presentinvention fills the long felt need of providing methods and means fordiagnosing, type-determination, treatment and prevention of infections,caused by coagulase negative bacteria.

SHORT DESCRIPTION OF THE FIGURES

The invention will be described in closer detail in the following, withsupport of the enclosed examples and figures, in which

FIG. 1 shows the adherence values as a function of fibrinogen coatingconcentration for the S. epidermidis strains 2, 19 and JW27 (Example1A),

FIG. 2 shows percent inhibition for antibodies against fibrinogen,compared to antibodies against fibronectin (Example 1B),

FIG. 3 shows percent inhibition as a function of competing fibrinogenconcentration (Example 1C),

FIG. 4 shows the protease sensitivity of adherence to fibrinogen(Example 1D).

FIG. 5 shows the inhibition of adherence by LiCl extract (Example 1E),

FIGS. 6A–6E show the complete nucleotide sequence of the fig gene fromS. epidermidis strain HB and the deduced amino acid sequence of theencoded protein (SEQ ID NOS:14 and 15). A putative ribosomal bindingsite (RBS) is underlined and a possible transcription terminationhairpin loop is double underlined. A putative signal sequence (S) isindicated with an arrow and the translational stop codon with anasterix. The start of the non-repetitive N-terminal region called A,harbouring the fibrinogen binding activity is indicated by an arrow. Rindicates the highly repetitive region. The 5 amino acid motif involvedin cell wall anchoring is indicated in bold characters and themembrane-spanning region is marked M (Example 3),

FIG. 7 shows a schematic drawing comparing the fibrinogen bindingprotein FIG of S. epidermis and the clumping factor (ClfA) of S. eureus.The similarity, (%), of corresponding regions in the proteins isindicated in the figure between the two protein bars. S is the signalsequence; A, the non-repetitive region harbouring the fibrinogen bindingactivity; R, the diamino acid residue repeat region; W the regionproposed to be involved in cell wall anchoring and M, the transmembranedomain. The numbers indicated refer to the amino acid positions inrespective proteins as shown in FIGS. 6A–6E and 7 and in reference(McDevitt et al., 1994) (Example 3),

FIG. 8 shows how GST-FIG fusion protein is captured to fibrinogen in adose dependent way (Example 10),

FIG. 9 shows the decrease of bacterial binding as a function of GST-FIGfusion protein, GST or FIG (Example 11),

FIG. 10 shows the relative adherence as function of serum dilution fortwo pre immune sera and a serum against GST-FIG and FIG, respectively(Example 12), and

FIG. 11 shows the relative bacterial adherence as a function of serumdilution for, on one hand, pre immune serum and, on the other hand,serum against GST-FIG (Example 12).

DESCRIPTION OF THE INVENTION

The present invention relates to a recombinant DNA molecule comprising anucleotide sequence, which codes for a protein or polypeptide havingfibrinogen-binding activity. The natural source of this nucleotidesequence is of course the S. epidermidis strain HB but with theknowledge of the nucleotide and deduced amino acid sequence presentedhere, the gene or parts of the gene can be isolated or madesynthetically. In particular the knowledge of the deduced amino acidsequence for the part of the protein responsible for the fibrinogenbinding activity can be used to produce synthetic polypeptides, whichretain or inhibit the fibrinogen binding. These polypeptides can belabeled with various compounds such as enzymes, fluorescence, biotin (orderivatives of), radioactivity, etc and used e.g. in diagnostic testssuch as ELISA- or RIA-techniques.

For production of a recombinant DNA molecule according to the inventiona suitable cloning vehicle or vector, for example a phagemid, plasmid orphage DNA, may be cleaved with the aid of a restriction enzyme whereuponthe DNA sequence coding for the desired protein or polypeptide isinserted into the cleavage site to form the recombinant DNA molecule.This general procedure is well known to a skilled person, and varioustechniques for cleaving and ligating DNA sequences have been describedin the literature (see for instance U.S. Pat. No. 4,237,224; Ausubel etal 1991; Sambrook et al 1989). Nevertheless, to the present inventors'knowledge, these techniques have not been used for the present purpose.If the S. epidermidis strain HB is used as the source of the desirednucleotide sequence it is possible to isolate said sequence and tointroduce it into a suitable vector in manner such as described in theexperimental part below or, since the nucleotide sequence is presentedhere, use a polymerase chain reaction (PCR)-technique to obtain thecomplete or fragments of the fig gene.

Hosts that may be used are, micro-organisms (which can be made toproduce the protein or active fragments thereof), which may comprisebacterial hosts such as strains of e.g. Escherichia coli, Bacillussubtilis, Staphylococcus sp., Lactobacillus sp. and furthermore yeastsand other eukaryotic cells in culture. To obtain maximum expression,regulatory elements such as promoters and ribosome binding sequences maybe varied in a manner known per se. The protein or active peptidethereof can be produced intra- or extra-cellularly. To obtain goodsecretion in various bacterial systems different signal peptides couldbe used. To facilitate purification and/or detection the protein orfragment thereof could be fused to an affinity handle and/or enzyme.This can be done on both genetic and protein level. To modify thefeatures of the protein or polypeptide thereof the gene or parts of thegene can be modified using e.g. in vitro mutagenesis; or by fusion ofother nucleotide sequences that encode polypeptides result in a fusionprotein with new features.

The invention thus comprises recombinant DNA molecules containing anucleotide sequence, which codes for a protein or polypeptide havingfibrinogen-binding properties. Furthermore the invention comprisesvectors such as e.g. plasmids and phages containing such a nucleotidesequence, and organisms, especially bacteria as e.g. strains of E. coli,B. subtilis and Staphylococcus sp., into which such a vector has beenintroduced. Alternatively, such a nucleotide sequence may be integratedinto the natural genome of the micro-organism.

The application furthermore relates to methods for production of aprotein or polypeptide having the fibrinogen binding activity of proteinFIG or active fragments thereof. According to this method, amicro-organism as set forth above is cultured in a suitable medium,whereupon the rusultant product is isolated by some separating method,for example ion exchange chromatography or by means of affinitychromatography with the aid of fibrinogen bound to an insoluble carrier.

Vectors, especially plasmids, which contain the protein FIG encodingnucleotide sequence or parts thereof may advantageously be provided witha readily cleavable restriction site by means of which a nucleotidesequence, that codes for another product, can be fused to the proteinFIG encoding nucleotide sequence, in order to express a so called fusionprotein. The fusion protein may be isolated by a procedure utilising itscapacity of binding to fibrinogen, whereupon the other component of thesystem may if desired be liberated from the fusion protein. Thistechnique has been described at length in WO 84/03103 in respect of theprotein A system and is applicable also in the present context in ananalogous manner. The fusion strategy may also be used to modify,increase or change the fibrinogen binding activity of protein FIG (orpart thereof) by fusion of other fibrinogen binding molecules.

The present invention also applies to the field of biotechnology thatconcerns the use of bacterial cell surface components as immunogens forvaccination against CNS infections. Immunisation using whole bacteriawill always trigger a highly polyclonal immunresponse with a low levelof antibodies against a given antigenic determinant. It is thereforpreferable to use the protein, polypeptide or DNA according to thepresent invention for immunisation therapies. Notably, immunisationtherapies can be conducted as so called passive and active immunisation.Passive immunisation using the inventive protein or DNA involves theraising of antibodies against the said protein or protein encoded by theadministered DNA in a suitable host animal, preferably a mammal, e.g. ahealthy blood donor or a cow, collecting and administering saidantibodies to a patient. One preferred embodiment is passiveimmunisation of a patient prior to surgery, e.g. operations involvingforeign implants in the body. Active immunisation using the inventiveprotein or DNA involves the administration of the said protein or DNA toa patient, preferably in combination with a pharmaceutically suitableimmunostimulating agent. Examples of such agents include, but are notlimited to the following: cholera toxin and/or derivatives thereof heatlabile toxins, such as E. coli toxin and similar agents. The compositionaccording to the present invention can further include conventional andpharmaceutically acceptable adjuvants, well known to a person skilled inthe art of immunisation therapy. Preferably, in an immunisation therapyusing the inventive DNA or fractions thereof, said DNA is preferablyadministered intramuscularly, whereby said DNA is incorporated insuitable plasmide carriers. An additional gene or genes encoding asuitable immunostimulating agent can preferably be incorporated in thesame plasmide.

Said immunisation therapies are not restricted to the above-describedroutes of administration, but can naturally be adapted to any one of thefollowing routes of administration: oral, nasal, subcutaneous andintramuscular. Especially the oral and nasal methods of administrationare potentially very promising, in particular for large-scaleimmunisations.

EXAMPLES

Starting Materials

Bacterial Strains, Phages and Cloning Vectors

Staphylococcus epidermidis strain HB was obtained from Dr Åsa Ljungh,Lund, Sweden.

E. coli strain TG1 and strain MC1061 were used as bacterial host forconstruction of the library and production of the phage stocks. The E.coli phage R408 (Promega, Madison, Wis., U.S.A.) was used as helperphage.

The phagemid vector pG8H6 used is described Jacobsson and Frykberg(1996).

All strains and plasmid- or phagemid-constructs used in the examples areavailable at the Department of Microbiology at the Swedish University ofAgricultural Sciences, Uppsala, Sweden.

Buffers and Media

E. coli was grown on LB (Luria Bertani broth) agar plates or in LB broth(Sambrook et al 1989) at 37° C. In appropriate cases the LB medium wassupplemented with glucose to a final conc. of 2%. Ampicillin was inappropriate cases added to the E. coli growth media to a final conc. of50 μg/ml. Staphylococci were grown at 37° C. on blood agar-plates(containing 5% final conc. bovine blood) or in Tryptone Soya Broth (TSBobtained from Oxoid, Ltd Basingstoke, Hants., England) PBS: 0.05M sodiumphosphate pH 7.1, 0.9% NaCl. PBS-T: PBS supplemented with TWEEN 20 to afinal conc. of 0.05%.

Preparation of DNA from Staphylococci and Streptococci

Strains of S. epidermidis or S. aureus were grown overnight in TSB. Nextmorning the cells were harvested and the chromosomal DNA preparedaccording to Löfdahl et al (1983). Chromosomal DNA from streptococci hasearlier been described in WO 95/07300.

Proteins and Other Reagents

Human fibrinogen was obtained from (IMCO Ltd, Stockholm, Sweden). Humanserum albumin (HSA), fibronectin IgA, lactoferrin and transferrin wereobtained from Sigma, St. Louis, U.S.A.). Bovine serum albumin (fractionV, ria grade) was obtained from USB (cat no.10868). α₂macroglobulin(α₂M) and collagen type I were obtained from Boehringer, Mannheim,Germany). Vitronectin was obtained from Bional, Tartu, Estonia and humanIgG from Kabi Stockholm, Sweden. Elastin was obtained from ICNPharmaceuticals Inc. CA, U.S.A. and pepsin from KEBO LAB, Stockholm,Sweden.

DNA probes were labelled with α³²P-ATP by a random-priming method(Multiprime DNA labelling system; Amersham Inc, Amersham, England)

Nitrocellulose (NC) filters (Schleicher & Schüll, Dassel, Germany) wereused to bind DNA in hybridisation experiments or proteins inWestern-blot techniques.

In order to analyse protein samples by native or sodium dodecylsulphate-polyacrylamid gel electrophoresis (SDS-PAGE) the PHAST-systemobtained from Pharmacia LKB Biotechnology, Uppsala, Sweden was usedaccording to the supplier's recommendations.

Oligonucleotides used were synthesised by Pharmacia (Uppsala, Sweden).

Micro Well plates (MaxiSorp, Nunc, Copenhagen, Denmark) were used in thepanning experiment. Plasmid DNA was prepared using Wizard Minipreps(Promega) and the sequence of the inserts was determined as described byJacobsson and Frykberg (1995). The sequences obtained were analysedusing the PC-gene program (Intelligenetics, Mountain View, Calif.,U.S.A.)

Routine Methods

Methods used routinely in molecular biology are not described such asrestriction of DNA with endonucleases, ligation of DNA fragments,plasmid purification etc since these methods can be found in commonlyused manuals (Sambrook et al., 1989, Ausubel et al., 1991). Ligationreactions were performed using Ready-To-Go T4 DNA Ligase (Pharmacia,Uppsala, Sweden). For polymerase chain reaction amplification the GeneAmp™ kit, obtained from Perkin Elmer Cetus, was used. Sequence reactionswere performed using “Sequence, version 2.0” kit (United StatesBiochemical Corporation, Cleveland, Ohio, U.S.A.). Alternatively the ABIPRISM Dye Terminator Cycle Sequencing Ready Reaction Kit was used andthe samples analysed using the Applied Biosystems 373A DNA Sequencer.

Example 1 The Adherence of Staphylococcus epidermidis to ImmobilisedFibrinogen and Investigation of the Nature of the Binding Mechanism(A–E)

Strains of Staphylococcus epidermidis isolated from cases of peritonitiswere grown on Blood agar plates at 37° C. overnight. The bacteria fromone plate was harvested with 5 ml phosphate buffered saline (PBS),washed once, and the optical density (OD) was adjusted to 1.0.

(A) Bacterial Adherence

Fibrinogen was dissolved in PBS at 10 mg/ml and added in serial 3-folddilution to microtiter wells (Nunc), from top to bottom. THe plates wereincubated overnight at room temperature (RT). To cover uncoated plasticsites the plates were coated with 2% bovine serum albumin for 1 hour at37° C. The plates were washed with PBS with 0.05% TWEEN 20 (PBST). Next,bacteria were added in serial 2-fold dilution in PBST, from left toright, to the fibrinogen coated microtiter plates. Bacterial adherencewas allowed for 2 hours at 37° C. or at 4° C. overnight. Non-adherentbacteria were washed off and the bound bacteria were air-dried. Thecrosswise dilution of both fibrinogen and bacteria allows estimation ofbacterial binding both as a function of fibrinogen concentration and ofamount of bacteria. Determination of bacterial adherence was done byoptical reading using a microtiter plate reader at A405. The turbidityand light scatter caused by bound bacteria results in a reading rangingfrom 0.00 to 0.20. An example of adherence values as a function offibrinogen coating concentration is shown in FIG. 1 for three differentstrains (2, 19 and JW27). These conditions for adherence determinationwere used in the following experiments.

(B) Adherence Blocking by Antibodies Against Fibrinogen

In a modification of the experiment performed above, antibodies againstfibrinogen (anti Fg) (Sigma) were added 1 hour prior to addition ofbacteria (OD=1.0) to the immobilised fibrinogen. As a control,antibodies against fibronectin (anti Fn) (Sigma) were added in aseparate experiment. FIG. 2 shows that antibodies against fibrinogen(circles) inhibited adherence better than antibodies against fibronectincould (squares). The mean values and standard errors from three separateexperiments are shown.

(C) Adherence Blocking by Soluble Fibrinogen

Soluble fibrinogen was added to the bacteria at concentrations indicatedin FIG. 3 and incubated for 1 hour at 37° C. before addition to platescoated with fibrinogen as described above. Adherence of S. epidermidisstrain 19 (filled circles) was inhibited to around 30%. As a control,inhibition of Staphylococcus aureus strain Newman was measured in asimilar experimental set-up (open circles). Mean values and standarderrors from three separate experiments are shown. Although significantinhibition of adherence of S. epidermidis was obtained, inhibition of S.aureus was more pronounced.

(D) Reduction of Binding After Protease Treatment of Bacteria

Bacteria were treated for 30 minutes at 37° C. with protease K, atconcentrations indicated in FIG. 4, prior to addition to immobilisedfibrinogen. Protease treated bacteria were extensively washed afterprotease treatment to avoid protease digestion of the immobilisedfibrinogen. Four different strains of S. epidermidis (2, 19, 269 and HB)and S aureus (strain Newman) were used in this experiment. All strainstested showed sensitivity to protease treatment; thus the adherence tofibrinogen depends on a surface protein.

(E) Adherence Blocking by LiCl Extract of S. epidermidis

S. epidermidis cells, grown and harvested as described above, weretreated with 1M LiCl at 40° C. for 2 hours with continuous gentlestirring. The bacteria were centrifuged and the bacteria-freesupernatant was filtered and dialysed against PBS. Surface associatedproteins bound to the cells by hydrophobic interactions are therebyreleased. This LiCl extract, presumably containing a fibrinogen bindingprotein, was used to inhibit adherence of S. epidermidis to immobilisedfibrinogen in the following way: LiCl extract at various dilutions wasadded to the immobilised fibrinogen and incubated for 1 hour at 37° C.The plates were washed and bacteria added for adhesion testing. FIG. 5shows that adherence was better the more the LiCl extract was diluted;i.e. an adhesion-inhibitory compound is present in the LiCl extract. Twoindependent experiments are shown.

Example 2 Isolation of a Clone Expressing Fibrinogen Binding Activity

A gene library of S. epidermidis strain HB was produced in a manner asdescribed by Jacobsson and Frykberg (1996). Staphylococcal DNA wasrandomly fragmented by sonication. The library resulted in 4×10⁷independent clones, which after amplification had a titer of 2×10¹⁰cfu/ml. Two hundred microlitres of the library were added to each ofthree fibrinogen coated wells and incubated for 4 hour at roomtemperature (RT). The wells were washed extensively with PBS-T and oncewith 50 mM Na-citrate/140 mM NaCl, pH 5.4. Finally, the bound phageswere eluted stepwise in the same buffer with decreasing pH (3.4 and1.8). The eluates from the three wells were neutralised with 2 MTris-HCl, pH 8.6. Aliquots of the eluates were used to infect E. coliTG1 cells, which thereafter were grown overnight on LA plates containingglucose and ampicillin. The colonies (obtained after infection of TG1cells with the phage and eluted at pH 3.4 and 1.8 in the primarypanning) were collected by resuspension in LB medium and infected withhelper phage R408 [10¹⁰ plaque-forming units (pfu)] for production ofenriched phage stocks. Thereafter, the infected bacteria were mixed with4 ml 0.5% soft agar and poured on one LA plate with ampicillin. Afterincubation over night 37° C. the phages were collected as described byJacobsson and Frykberg (1996). The resulting phage stock was repannedagainst fibrinogen as described above. The result presented in Table 1.shows there is an enrichment of clones having affinity to fibrinogen.

TABLE 1 Ligand Panning Fibrinogen IgG 1st Wash 1.6 × 10³ cfu/ml — pH 5.41.6 × 10³ cfu/ml — pH 3.4 2.1 × 10³ cfu/ml — pH 1.8 7.0 × 10³ cfu/ml —2nd Wash 1.2 × 10³ cfu/ml 2.2 × 10² cfu/ml pH 5.4 4.4 × 10³ cfu/ml 6.2 ×10² cfu/ml pH 3.4 4.3 × 10⁴ cfu/ml 1.4 × 10³ cfu/ml pH 1.8 2.0 × 10³cfu/ml 8.0 × 10² cfu/ml

Example 3 DNA Sequencing and Sequence Analysis

Eight colonies coming from the second panning (pH 3.4) againstfibrinogen described in Example 2 were chosen for further studies.Phagemid DNA from these colonies was prepared and partially sequenced.Seven of the clones seemed to contain the same insert. One of theseseven clones called pSE100 was chosen for further studies. Purifiedphagemid DNA from the clone pSE100 was analysed by restriction mappingwhich revealed that the phagemid contained an insert of ˜1.8 kilo basepair (kb). The nucleotide (nt) sequences of the complete inserts ofpSE100 were determined and the nt and deduced amino acid (aa) sequenceswere analysed using the PC-gene program. This analysis revealed that theinsert of pSE100 contains an open reading frame of 1.745 nt (sequencelist). Thus the insert encodes a 582 aa protein, termed protein FIG (andthe corresponding gene termed fig), with a calculated molecular mass of˜65 kDa (sequence list). Furthermore, the sequence analysis show thatthe insert of pSE100 is in the correct reading frame with the vectorsequences in the 5′- and 3′-ends. This means that the insert gives riseto a fusion with the pel leader and the myc tail (sequence list) andthat the native 5′- and 3′-ends of the fig gene is not present in thepSE100 clone.

To obtain the missing 5′ and 3′ end of the fig gene a Southern blotanalysis was performed using chromosomal DNA from strain HB digestedwith various restriction enzymes. The probe was prepared as follows; twooligonucleotides (5′CAACAACCATCTCACACAAC3′ which is SEQ ID NO:1 and5′CATCAAATTGATATTTCCCATC3′ which is SEQ ID NO:2) were used to PCRamplify a ˜1.3 kb fragment from the insert of pSE100. The PCR generatedfragments were 32P-labelled using random priming. After hybridisationusing stringent conditions the NC-filter was washed and subjected toautoradiography. The result showed that the Xbal cleavage gave a singleband in size of ˜6 kb. The corresponding fragment was subsequentlyligated into Xbal digested pUC18 vector. After transformation clonesharbouring the ˜6 kb Xbal-fragment were identified by colonyhybridisation using the same probe as in the Southern blot experiment.One such clone, called pSE101 was chosen for further studies. DNAsequence analysis showed that the fig gene consist of an open readingframe of a 3291 nt, encoding a protein, called FIG of 1097 aa with acalculated molecular mass of ˜119 kDa (FIGS. 6A–6E). The FIG proteinconsist of several typical features found among Gram-positive cellsurface bound proteins, like a N-terminal signal sequence and aC-terminal 5 amino acid motif (indicated in bold characters, followed bya stretch of 17 hydrophobic aa ending in a stretch of charged aa (FIG.6). Following the signal sequence, there is a region, called A of 773aa. The insert of pSE100 contains the sequence corresponding to residue75 to 656 of the A region (FIG. 7). The A region is followed by a highlyrepetitive region of 216 aa composed of tandemly repeated aspartic acidand serine residues, called R (FIGS. 6A–6E and 7). The dipeptid regionconsist of an 18 bp sequence unit (consensus of GAX TCX GAX TCX GAX AGXwhich is SEQ ID NO:3) repeated 36 times. The 18 bp sequence is almostmaintained perfect throughout the whole R region except for the secondunit which is truncated, consisting of only 12 of the 18 bp and the 3′end of the region where the consensus sequence is slightly disrupted(units 32, 34 and 36). The changes in the later units also result in anamino acid exchange which disrupt the DS repeat.

Using the deduced amino acid sequence of protein FIG protein databaseswere screened for sequence similarities. Interestingly, the searchshowed that the highest score obtained was for the clumping factor(ClfA) of S. aureus (FIG. 7). This protein binds fibrinogen and has beenshown to promote aggregation of bacteria in the present of plasma.Beside similarities in the N- and C-terminal part encoding the signalsequence and the cell membrane spanning domain, respectively the mostobvious similarity with the clumping factor is the repetitive R region.In both ClfA and FIG protein, the DS repeat region is encoded by thesame 18 bp consensus unit. Comparing the nucleotide sequences of fig andclfA shows that the R regions have an extensive homology. In addition,protein FIG also shows homology to ClfA m the A region, thenon-repetitive fibrinogen binding domain (FIG. 7).

Example 4 Properties of the Fibrinogen Binding Protein Encoded frompSE100

A) Specificity of the Fibrinogen Binding

The phagemid pSE100 was electroporated into competent E. coli TG1 cells.After growth over night on a LA plate (containing ampicillin andglucose) one colony containing pSE100 was grown over night and infectedwith the helper phage R408 for production of an enriched phase stock.The resulting phage stock containing recombinant phages expressing theinsert of pSE100 had a titer of 3×10⁹ cfu/ml. The phage stock of pSE100was used to pan against 13 different proteins coated in microtiter wellsand to one uncoated well. To each well containing the respective protein(or to the uncoated well) 200 μl of the phage stock of pSE100 was added.After panning for three hours at RT under gentle agitation the wellswere washed extensively, using PBST and a sample of the last wash wascollected. The bound phages were eluted with Na-Citrate buffer pH 1.8.The eluted samples were immediately neutralised using 1M Tris-HCl pH8.6. The eluted phages and the phages from the wash were allowed toseparately infect E. coli TG1 cells and after infection, the cells wereplated on LA plates containing ampicillin and glucose. The plates wereincubated over night at 37° C. and the frequency of colonies wascounted. The result of this experiment is presented in Table 2 whichshows the fibrinogen binding specificity of the protein expressed bypSE100.

TABLE 2 Ligand Wash Eluate pH 1.8 Fibrinogen 1.1 × 10⁴ cfu/ml 1.4 × 10⁷cfu/ml α₂M 2.0 × 10² cfu/ml 2.0 × 10³ cfu/ml BSA <10² cfu/ml 8.0 × 10²cfu/ml Collagen type I 6.0 × 10² cfu/ml 1.2 × 10³ cfu/ml Elastin 8.0 ×10² cfu/ml 5.2 × 10³ cfu/ml Fibronectin 6.0 × 10² cfu/ml 2.4 × 10⁴cfu/ml HSA 8.0 × 10² cfu/ml 2.2 × 10³ cfu/ml IgA 6.0 × 10² cfu/ml 6.8 ×10⁴ cfu/ml IgG 4.0 × 10² cfu/ml 4.4 × 10³ cfu/ml Lactoferrin 6.0 × 10²cfu/ml 8.2 × 10³ cfu/ml Pepsin 1.8 × 10² cfu/ml 3.7 × 10⁴ cfu/mlTransferrin 2.0 × 10² cfu/ml 2.4 × 10³ cfu/ml Vitronectin <10² cfu/ml2.2 × 10³ cfu/ml Plastic 2.4 × 10³ cfu/ml 9.0 × 10³ cfu/ml(B) Inhibition Experiment

The pSE100 phage stock was diluted to a titer of ˜5×10⁶ cfu/ml. Of thisphage solution samples (180 μl) were taken and separately incubated forone hour with different concentrations of fibrinogen, BSA or IgG beforetransferred to fibrinogen coated microtiter wells. After panning forthree hours at RT under gentle agitation, the wells were washedextensively using PBST. The bound phages were eluted with Na-Citratebuffer pH 1.8. The eluted samples were immediately neutralised using 1MTris-HCl pH 8.6. The eluted phages were allowed to infect E. coli TG1cells and after infection, the cells were plated on LA plates containingampicillin and glucose. The plates were incubated over night at 37° C.and the frequency of colonies was counted. The result of this experimentis presented in Table 3, which shows that the binding to fibrinogen isinhibited by fibrinogen but not with the other tested proteins.

TABLE 3 Conc. of different Soluble ligands ligands (μg/ml) FibrinogenBSA IgG 0 7.6 × 10⁴ cfu/ml 7.6 × 10⁴ cfu/ml 7.6 × 10⁴ cfu/ml 0.1 4.4 ×10⁴ cfu/ml 7.0 × 10⁴ cfu/ml 6.2 × 10⁴ cfu/ml 1 3.6 × 10⁴ cfu/ml 9.3 ×10⁴ cfu/ml 9.0 × 10⁴ cfu/ml 10 1.5 × 10⁴ cfu/ml 6.3 × 10⁴ cfu/ml 7.8 ×10⁴ cfu/ml 100 3.8 × 10³ cfu/ml 6.4 × 10⁴ cfu/ml 7.3 × 10⁴ cfu/ml 10003.0 × 10² cfu/ml 6.9 × 10⁴ cfu/ml 7.6 × 10⁴ cfu/ml

Example 5 Western Blot Experiment

E. coil cells of strain TG1 and MC1061 containing pSE100 were grown inLB (containing ampicillin and glucose) over night at 37° C. The nextmorning the cells were harvested by centrifugation, resuspended in LB(containing ampicilin, glucose and 0.1 M IPTG and further incubated at37° C. Twelve hours later the cells were harvested by centrifugation andboth the cells and the supernatant were taken care of. Four volumes ofacetone were added to the supernatant and the resulting precipitate wascollected by centrifugation, air-dried and resuspended in ice-cold PBS.Prior to electrophoresis the cells and the precipitate from thesupernatant were resuspended separately in a sample buffer containing2.5% SDS and 5% beta-mercaptoethanol and boiled for two minutes. Afterdenaturation the samples were analysed run under reducing conditionsusing the PHAST-system (Pharmacia) on a 8–25% gradient gel usingSDS-buffer strips. After the electrophoresis was completed a NC-filterpreviously soaked in PBS was put on the gel and the temperature raisedto 45° C. After ˜45 minutes the NC-filter was wetted with 1 ml PBS,gently removed and placed in 15 ml PBS containing 0.1% TWEEN 20 solution(PBST 0.1%) for 30 minutes in RT (under gentle agitation and with twochanges of PBST 0.1% solution). After the last change of PBST 0.1%fibrinogen was added to a final conc. of 20 ng/ml and the filter wasincubated for four hours at RT under gentle agitation. The filter wassubsequently washed for 3×10 minutes using PBST 0.1% and HRP-conjugatedrabbit anti-human fibrinogen antibodies (DAKO code A 080, diluted 1:500in PBST 0.1%) were added and the filter was incubated for 1 hour at RTunder gentle agitation. After washing the filter 3×10 minutes using PBST0.1% the bound fibrinogen was visualised by transferring the filter to asolution containing a substrate for the horse radish peroxidase (6 ml4-chloro-1-naphtol (3 mg/ml in methanol)+25 ml PBS+20 μl H₂O₂). Theresult showed that a fibrinogen binding protein was found in both typesof samples (cells and growth media) in both E. coil cells harbouringpSE100, while no such protein was found in the control cultures of E.coil TG1 and MC1061. The fibrinogen binding protein expressed from thepSE100 was in the approximate size as expected from the deduced aminoacid.

Example 6 The Occurrence of the Fig Gene and the Use of Fig Gene toIdentify S. epidermidis in Diagnostic Test

Purified chromosomal DNA from S. aureus strain 8325-4, Streptococcusequi subsp. equi strain 196 and subspecies zooepidemicus strain Z5,Streptococcus pyogenes strain 2–1047, Streptococcus dysgalactiae strain8215 were cleaved using the restriction enzyme EcoRI. The cleavedsamples were run on an 0.8% agarose-gel together with chromosomal DNAfrom S. epidermidis strain HB cleaved with various restriction enzymes.After the electrophoresis was completed, the separated DNA fragmentswere transferred to a NC-filter using the Vacuum blotting system fromPharmacia. After the transfer the filter was hybridised under stringentconditions (in a solution containing 6× SSC, 5× Denhart, 0.5% SDS at 65°C.) using a probe designed based on the nucleotide sequence of theinsert of pSE100. This probe had earlier been prepared as follows, twooligonucleotides: (5′-AGGTCAAGGACAAGGTGAC-3′ which is SEQ ID NO:4 and5′-CAACAACCATCTCAC ACAAC-3′ which is SEQ ID NO:1) were ordered(Pharmacia) and used as a primer pair in a PCR (25 cycles of 94° C. 1minute, 50° C. 30 seconds, 72° C. 1 minute using an Perkin Elmer CetusThermal Cycler 480) to amplify an ˜150 bp fragment of the insert ofpSE100. The amplified material was run on an agarose gel and the ˜150 bpfragment was purified and radioactively labelled using ³²P-dATP and theMultiprime DNA labelling system (Amersham). The filter was hybridisedover night and subsequently washed in a washing solution (0.2% SSC, 0.1%SDS) at 60° C. and autoradiographed. The result showed that nohybridisation was detected in the samples originating from streptococciand S. aureus while hybridisation occurred to the samples coming fromthe S. epidermidis strain HB.

To investigate the occurrence of the fig gene in other strains of S.epidermidis the following PCR reaction was set up. Chromosomal DNA from13 different clinical isolates of S. epidermidis was used as templates.The same primers and the same PCR conditions as described above wereused. The result showed that an amplified product of ˜150 bp could bedetected (using a 2% agarose gel) in all strains of S. epidermidis butnot in the control samples original containing chromosomal DNA from S.aureus and S. pyogenes.

Example 7 A PCR Amplification Assay for Analysis of Corresponding DSRepeat Regions from Various Isolates of S. epidermidis

McDevitt and Foster (Microbiology, 1995, 141:937–943) have shown thatthe DS repeat region in various isolates of S. aureus strains may differconsiderable. To investigate if the DS repeat region in S. epidermidisalso varies in size between different isolates following experiment wasperformed. A pair of primers (5′CCGATGAAAATGGAAAGTATC3′ which is SEQ IDNO:5 and 5′TCCGTTATCTATACTAAAGTC3′ which is SEQ ID NO:6) hybridising onthe 5′ and 3′ side, respectively, of the DS repeat region of protein FIGwere used to PCR amplify the corresponding region in 11 differentisolates of S. epidermidis. The amplification was performed as follows,after initial denaturation for 1 mm. at 95° C. a cycle started with adenaturation step for 30 sec. at 95° C., followed by an annealing timeof 1 mm at 50° C. and a elongation period of 2 mm. at 72° C. The cyclewas repeated 25 times and ended in an final elongation period of 7 mm.at 72° C. The PCR products representing the DS region of respectivestrain were analysed by agarose-gel electrophoresis. The result showedthat one band of various length was present in each sample. Theconclusion from this is that this type of method can be used as adiagnostic test to get a “fingerprint” of a particular strain. Thismight be useful in e. g. tracing a the origin of an infection.

Example 8 The Use of the DS Fragment of Strain HB to Identify OtherHomologous Genes in Coagulase-positive and -negative Staphylococci

A DNA fragment consisting of the DS repeat region was constructed asfollows. One pair of oligonucleotide primers (5′ACTGATCATGATGACTTTAGT3′which is SEQ ID NO:7 and 5′TCCGTTATCTATACTAAAGTC3′ which is SEQ ID NO:6)was used to PCR amplify the DS region of strain HB using the sameconditions as described above. The amplification resulted in a ˜700 bpfragment which was radioactively (³²P) labelled using random priming.This probe was used in a Southern blot analysis using chromosomal DNA(cleaved with EcoRI) from various species of staphylococci (S. aureus,S. epidermidis strain HB, S. haemolyticus strain 789 and strain SM131,S. lugdunensis, S. schleiferi, S. intermedius, S. lentus, S. sciuri, S.carnosus, S. saprophyticus and S. hyicus.

The hybridisation was performed under stringent conditions at 65° C.over night. The next day the filter was washed at 65° C., using 2× SSCfollowing autoradiography. The result showed that at least one specificband was present for the following species; S. aureus, S. epidermidisstrain HB, S. haemolyticus strain 789 and strain SM131, S. lugdunensis,S. intermedius, S. sciuri, S. carnosus (weak signal) and S. hyicus. Thisresult shows, that it is possible to clone and identify thecorresponding regions in these species.

Example 9 Production of GST-FIG

By polymerise chain reaction, a DNA fragment was amplified encoding aportion of the fibrinogen binding protein. Upper primer wasGCGGATCCAATCAGTCAATAAACACCGACGAT (SEQ ID NO:8) and lower primer wasCGGAATTCTGTTCGGACTGATTTGGAAGTTCC (SEQ ID NO:9). Amplification was donefor 30 cycles at 94° C. 30 seconds, 60° C. 30 seconds, 72° C. 2 minutesbeginning with 94° C. for 4 minutes and ending with 72° C. for 4minutes. The amplified fragment was digested with EcoRI and Bam HI.Plasmid pGXT-4T (Pharmacia, Uppsala, Sweden)was digested with EcoRI andBam HI, mixed with the digested fragment and the mixture ligated usingT4 DNA ligase according to standard procedures. The ligated DNA wastransformed into E. coli strain TGI. A transformant was isolated with aplasmid encoding a fusion protein composed of glutathione thiotransferase and fibrinogen binding protein. The protein was purifiedwith the vector plasmid according to Pharmacia's instructions. Thepurified GST-FIG protein was subjected to Western affinity blot. It wasrun on polyacrylamide gel electrophoresis, transferred to nitrocellulosepaper by passive diffusion, the paper treated with fibrinogen (5 μg/ml)for 2 hours at room temperature, followed by rabbit anti fibrinogenantibodies conjugated to HRP. A band corresponding to a molecular weightof approx. 100 kDa was seen. Omitting fibrinogen in a control experimentdisplayed no band.

Example 10 Demonstration of Binding of GST-FIG to Stationary PhaseFibrinogen

Microtiter wells were coated with human fibrinogen (Sigma Chemicals Co.)at a concentration ranging from 2.5 to 20 μg/ml at room temperatureovernight. The plates were aftercoated with 2% bovine serum albumin(BSA) for one hour at 37° C. The microtiter plates were washed threetimes and GST-FIG was added to the wells at concentrations of 25, 50 or100 μg/ml (indicated by the three separate lines in FIG. 8) and theplates incubated for two hours at 37° C. Capture of GST-FIG to thefibrinogen layer was, after washing, detected by antibodies (diluted1000 times) raised in a rat a against His-FIG. Binding of antibodieswas, after washing, detected with rabbit anti rat IgG antibodiesconjugated with HRP. The substrate for HRP was OPD tablets (Dakopatts)with H₂O₂. Colour reaction was measured at 495 nm. FIG. 8 shows thatGST-FIG is captured to fibrinogen in a dose dependent way.

Example 11 Inhibition of S. epidermidis Adherence to Fibrinogen by FIG

Fibrinogen at 2 μg/ml was used to coat microtiter wells overnight atroom temperature and aftercoated as above. GST-FIG fusion protein, GSTor FIG was added at concentrations indicated in FIG. 9. Radioactivelylabeled bacteria was added immediately after, and incubated at 37° C.for two hours. Decrease of bacterial binding as a function of GST-FIGfusion proteins GST or FIG is shown in FIG. 9. The symbols in FIG. 9 arethe following squares—inhibition by GST-FIG (mean and SE of fiveindependent experiments are shown); triangles—inhibition by GST carrierprotein; circles—inhibition by FIG after thrombin digestion. Only thefusion protein and FIG molecules could inhibit binding.

Radioactive labelling of bacteria was obtained by growing them in thepresence of tritiated thymidine (20 μCi/ml, specific activity 81Ci/mmole) for 5 hours in LB.

Cleavage of GST-FIG was achieved by adding thrombin and incubating at37° C. for 2 hours.

Example 12 Inhibition of S. epidermidis Adherence to Fibrinogen byAntibodies Against GST-FIG and FIG

Fibrinogen at 2 mg/ml was used to coat microtiterwells overnight at roomtemperature and aftercoated as above. Radiolabelled S. epidermidis wereincubated with different dilutions of sera for 1 hour at 37° C. Thebacteria—serum mixtures were then added to the wells and adherence wasallowed to take place for two hours at 37° C. Non adherent bacteria werewashed away and the amount of adherent bacteria were determined as inexample 11 above. Four serum samples were used: 1) Serum from beforeimmunisation from rat No 1. 2) Serum from before immunisation from ratNo 2. 3) Serum from rat No 1 immunised with GST-FIG. 4) Serum from ratNo 2 immunised with FIG generated by thrombin cleavage. From FIG. 10 itcan be seen that adherence is reduced after incubation with sera againstFIG or against the GST-FIG fusion protein. With relative adherence of1.0 is meant the adherence obtained after incubation of theradiolabelled bacteria with phosphate buffered saline.

The experiment was repeated, and data from adherence blocking using serataken before immunisation and serum taken after immunisation withGST-FIG is shown in FIG. 11.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

REFERENCES

-   Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D.,    Seidman, J. G., Smith, J. A. and Struhl. K. (eds.) (1991) Current    Protocols in Molecular Biology, Greene Publishing and    Wiley-Intersciences, New York.-   Bodén, M. K. and Flock, J.-I. (1995) J.Clin.Microbiol. 33:    2347–2352. Incidence of the highly conserved fib gene and expression    of the fibrinogen binding (Fib) protein among clinical isolates of    Staphylococcus aureus.-   Jacobsson, K. and Frykberg, L. (1995) Cloning of ligand-binding    domains of bacterial receptors by phage display. BioTechniques    18:878–885-   Jacobsson, K. and Frykberg, L. (1996) Phage display shotgun cloning    of ligand-binding domains of prokaryotic receptors approaches 100%    correct clones. BioTechniques 20:1070–1081.-   Löfdahl, S., Guss, B., Uhlén, M., Philipson, L. and    Lindberg, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:697–701.-   McDevitt, D., Francois, P., Vaudaux, P. and Foster, T. J. (1994)    Mol. Microbiol. 11:237–248.-   Moreillon, P., Entenza, J. M. Francioli, P., McDevitt, D.,    Foster, T. J., Francois, P. and Vaudaux, P. (1995) Infect. Immun.    63:4738–4743.-   Sambrook, J., Fritsh, E. F. and Manias, T. (1989) Molecular cloning,    A laboratory manual, second ed, Cold Spring Harbour Laboratory    Press, New York.-   Wadström, T., and Rozgony, F. (1986) Virulence determinants of    coagulase-negative staphylococci pp 123–130. In “Coagulase-negative    staphylococci” Eds. M{dot over (a)}rdh, P.-A. and Schleifer, K. H.-   Almquist & Wiksell International, Stockholm, Sweden. ISBN    91-22-00783-0.-   Patents or patent applications cited: WO 95/07300, U.S. Pat. No.    4,237,224, WO 84/03103.

The nucleotide sequence shown in the above SEQ ID NO: 10 encodes aprotein which contains 593 amino acids. SEQ ID NO: 11 is the amino acidsequence of this protein.

SEQ ID NO: 12 is the nucleotide sequence containing 1746 nitrogenousbases which code for the 582 amino acid FIG protein. As discussed above,the 582 amino acid FIG protein is encoded by the insert of pSE100. Thenucleotide sequence of SEQ ID NO: 12 corresponds to bases 255–2000 shownin FIGS. 6A–6E.

SEQ ID NO: 13 is the deduced amino acid sequence encoded by SEQ ID NO:12. Thus SEQ ID NO: 13 is the 582 amino acid sequence of the FIG proteinand thereby corresponds to amino acids 75–656 of the sequence depictedin FIGS. 6A–6E. In other words SEQ ID NO: 13 is the amino acid sequenceof SEQ ID NO: 11 without the Pel leader sequence and the Myc tail.

SEQ ID NO: 15 is the deduced amino acid sequence encoded by SEQ ID NO:14, i.e., the amino acid sequence shown in FIGS. 6A–6E.

1. An isolated antibody that binds to a Staphylococcus epidermidispolypeptide consisting of the amino acid sequence of SEQ ID NO:
 13. 2.The antibody of claim 1, wherein the polypeptide is further fused toglutathione-S-transferase (GST).
 3. The antibody of claim 1, wherein thepolypeptide is encoded by the nucleotide sequence of SEQ ID NO: 12.